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Proc. Nail. Acad. Sci. USA Vol. 89, pp. 10721-10725, November 1992 Pharmacology Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids (eicosanoids/lipids/oxidative stress/peroxidation/free radicals) JASON D. MORROW, JOSEPH A. AWAD, HOLLIS J. BOSS, IAN A. BLAIR, AND L. JACKSON ROBERTS II* Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN 37232.6602 Communicated by Philip Needleman, July 21, 1992 (receivedfor review March 11, 1992) ABSTRACT We recently reported the discovery ofa series the formation ofthese prostanoids occurs independent ofthe of bioactive prostaglandin F2-like compounds (F2-isoprostanes) catalytic activity of the cyclooxygenase enzyme, which had that are produced in vivo by free radical-catalyzed peroxidation been considered obligatory for endogenous prostanoid bio- ofarachidonic acid independent ofthe cyclooxygenase enzyme. synthesis. Circulating levels ofthese compounds were shown Inasmuch as phospholipids readily undergo peroxidation, we to increase dramatically in animal models of free radical examined the possibility that F2-isoprostanes may be formed in injury (8). Interestingly, the levels of these prostanoids in situ on phospholipids. Initial support for this hypothesis was normal human plasma and urine are one or two orders of obtained by the rmding that levels of free F2-isoprostanes magnitude higher than those of prostaglandins produced by measured after hydrolysis oflipids extracted from livers ofrats the cyclooxygenase enzyme. Formation ofthese compounds treated with CCI4 to induce lipid peroxidation were more than proceeds through intermediates composed of four positional 100-fold higher than levels in untreated animal. Further, peroxyl radical isomers of arachidonic acid which undergo increased levels of lipid-associated F2-isoprostanes in livers of endocyclization to yield bicyclic endoperoxide PGG2-like CCI4-treated rats preceded the appearance of free compounds compounds. The PGG2-like compounds are then reduced to in the circulation, suggesting that the free compounds arose PGF2-like compounds (9). In conjunction with the Committee from hydrolysis of peroxidized lipids. This concept was sup- of Eicosanoid Nomenclature constituted by the Joint Com- ported by demonstrating that free F2-isoprostanes were re- mission on Biochemical Nomenclature, a facile nomencla- leased after incubation of lipid extracts with bee venom phos- ture for the individual compounds produced by this mecha- pholipase A2 in vitro. When these lipid extracts were analyzed nism is being developed, based on the general term "iso- by HPLC, fractions that yielded large quantities of free F2- prostanes" and the type of prostane ring they contain. isoprostanes after hydrolysis eluted at a much more polar Unsaturated fatty acids in phospholipids readily undergo retention volume than nonoxidized phosphatidylcholine. Anal- peroxidation upon exposure to free radicals. Peroxidation of ysis of these polar lipids by fast atom bombardment mass arachidonoyl phospholipids would result in the formation of spectrometry established that they were F2-isoprostane- the positional peroxyl isomers of arachidonic acid that are containing species of phosphatidylcholine. Thus, unlike cy- intermediates leading to the formation of isoprostanes (9). clooxygenase-derived prostanoids, F2-isoprostanes are initially Therefore, we investigated the possibility that isoprostanes formed in situ on phospholipids, from which they are subse- may be formed completely in situ on phospholipids. quently released preformed, presumably by phospholipases. Molecular modeling of FI-isoprostane-containing phospholip- ids reveals them to be remarkably distorted molecules. Thus, EXPERIMENTAL PROCEDURES the formation of these phospholipid species in lipid bilayers Analysis of F2-Isoprostanes. Purification, derivatization, may contribute in an important way to alterations in fluidity and analysis ofF2-isoprostanes by gas chromatography/mass and integrity of cellular membranes, well-known sequelae of spectrometry were performed as described (8, 9). oxidant is ury. Animal Model of Free Radical-Induced Lipid Peroxidation. Endogenous lipid peroxidation was induced in rats by ad- Free radicals derived from oxygen are thought to play an ministration ofCC4 as described (10). At various time points, important role in the pathophysiology ofan increasingly wide animals were sacrificed and the livers were removed, frozen variety ofhuman diseases (1-4). Nonetheless, much remains in liquid N2, and either processed immediately or stored at to be understood about mechanisms ofoxidant injury in vivo. -70°C. We have previously determined that formation of Polyunsaturated fatty acid-containing lipids are recognized F2-isoprostanes by autoxidation of lipids in vitro is com- targets of oxidant damage, readily undergoing peroxidation pletely inhibited at -70°C. upon exposure to free radicals. Peroxidation of lipids can Extraction, Purification, and Hydrolysis of Lipids. Lipids greatly alter the physicochemical properties of membrane from livers were extracted (11), subjected to alkaline hydro- lipid bilayers, resulting in severe cellular dysfunction. In lysis, and subsequently analyzed for F2-isoprostanes as de- addition, a variety of lipid byproducts are produced as a scribed (9). Depending on the experiment, 0.005% butylated consequence of lipid peroxidation, some of which can exert hydroxytoluene or 0.5% triphenylphosphine was added to adverse biological effects (5-7). lipid extracts during the extraction procedure. Recently, we reported the discovery that a series of Release of F2rsoprostanes from Lipid Extracts by Apis prostaglandin (PG)-like compounds capable of exerting po- melifera Venom Phospholipase A2. Lipid extracts of livers of tent biological activity are produced in vivo in humans as CCL4-treated rats (containing approximately 1 ,mol of phos- products of free radical-catalyzed peroxidation of arachi- pholipid) were allowed to react with Apis mellifera venom donic acid (8). The notable aspect of this discovery was that phospholipase A2 (approximately 200 ,g) (Sigma) as de- scribed (12). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PG, prostaglandin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 10721 Downloaded by guest on September 29, 2021 10722 Pharmacology: Morrow et al. Proc. Natl. Acad Sci. USA 89 (1992) As a positive control for enzymatic activity, phosphatidyl- tanes measured after the hydrolysis of lipid extracts from choline containing [3H]arachidonate in the sn-2 position livers of rats 2 hr after administration of CCL1 (2 ml/kg) were (New England Nuclear) was added to the incubation mixture 825 + 198 ng/g of liver (n = 5). These levels were 142-fold and the percent of radiolabeled arachidonate released was higher than levels measured after saponification of lipid determined. After 2 hr, the diethyl ether was evaporated and extracts of livers of untreated rats (5.8 + 1.2 ng/g of liver) (n free arachidonic acid and phospholipids were extracted from = 5). Levels of free F2-isoprostanes measured in lipid ex- 1/2 of the incubation mixture at pH 7 with CHC13/methanol tracts that were not subjected to saponification were <2% of (2:1, vol/vol) (13). The organic layer was then evaporated levels measured after saponification. under a stream of N2 and the residue was subjected to TLC The following evidence was obtained confirming that the (silica gel 60A LK6D, Whatman) using a solvent system of compounds detected after alkaline hydrolysis of the lipid petroleum ether/diethyl ether/acetic acid (90:10:1, vol/vol). extracts of liver were identical to F2-isoprostanes previously Free arachidonic acid migrates with an Rf of 0.18 and identified in human plasma and urine: (i) analysis of the phospholipids remain at the origin. The ratio of free arachi- compounds by gas chromatography/mass spectrometry as a donic acid to esterified phospholipid arachidonate was de- deuteriated silyl ether derivative indicated the presence of termined by comparing the amount of radioactivity present three hydroxyl groups; (ii) analysis after catalytic hydroge- between Rf 0.15-0.25 to that present at the origin. nation indicated the presence of two double bonds; (iii) all Free F2-isoprostanes were extracted from the remaining 1/2 compounds formed cyclic boronate derivatives, indicating of the reaction mixture with an equal volume of ethyl acetate the presence of cis prostane ring hydroxyl groups; and finally twice after decreasing the pH to 3 with 1 M HCl. After (iv) analysis by electron ionization mass spectrometry re- evaporation of the organic layer under N2, the residue was vealed multiple compounds eluting from the capillary GC resuspended in 0.5 ml of methanol to which 10 ml of pH 3 column over approximately a 40-sec period that yielded mass water was added. Free F2-isoprostanes were then reextracted spectra essentially identical to those obtained previously for by using C18 Sep-paks (Waters), purified by TLC, and F2-isoprostanes (data not shown) (8, 9). quantified by mass spectrometry as described (8). The per- The above data were consistent with the hypothesis that cent of total F2-isoprostanes released by bee venom phos- F2-isoprostanes are present in vivo in ester linkages to lipids. pholipase A2 was calculated by determining the ratio of the However,

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